Chapter 2 - Clinic of membrane and bone graft substitute
Assistant Professor, University of Michigan Junro YAMASHITAI.

Introduction

In order to achieve an implant prosthesis that is both aesthetically and functionally agreeable, it isdesirable for the quantity and quality of the bone to be sufficient with soft tissues. An implant installation
to bone structures with such favorable conditions would result in a firm integration with the bone, andone harmonious co-existence with the surrounding gingivae structures and the neighboring natural teeth
would be possible. Unfortunately, these ideal cases are rare in the clinical settings, as the alveolar bone
resorption is induced upon tooth loss. In implant treatments conducted to replace the extracted tooth thathas resulted from periodontal disease damage, the alveolar bone is often found to have been resorbed bothvertically and horizontally and sufficient amount of bone is difficult to be obtained. The progress of bone
resorption that results from loss of teeth can rapidly occur within a short period of time in the regions
where the alveolar bone is thin (particularly the anterior region of both mandible and the maxilla). The
buccal alveolar bone is thinner in the alveolar region of the anterior teeth than the labial side. In case of
tooth loss the buccal bone resorption is often found to occur along with the vertical bone resorption
leading to a general collapse of the buccal side of the bone. In cases where such resorption of the bone has
affected the alveolar bone structure, the implant prosthesis with an aesthetic outcome can become
difficult to be achieved. These issues can be overcome by conducting alveolar bone augmentation
procedures in conjuction with the implant installation.
The reason for bone augmentation procedures to have widely spread as a dental clinical procedure can besaid to be due to the development of bone fillers and membrane materials. The rise in application of thismethod is also due to the synergistic effect with the increased need for the alveolar bone regeneration tobe established with high predictability. The scientific research for viable bone fillers that aid the boneregeneration are a subject of high interest where a significant number of materials have been developed
and are applied in the clinical settings. The main subject of current research lies in the development ofcell therapy with bone fillers combined with growth factor agents and application of stem cells. With
regards to the bone fillers with growth factor agents, there are some that have already been licensed foruse in field of dental surgery. Unfortunately, the majority of these bone fillers and membranes have notyet been approved under the Pharmaceutical Affaires Law in Japan, therefore cannot be used in thedental clinical settings in this country. For this reason, there is a general lack of knowledge and difficultyin gaining updated information on bone regeneration therapy at present, and are progressively becoming
left behind from the global clinical advancements, reminiscent of national isolation period. I still believethat sufficient understanding of the forefront of clinical sciences is essential in order to provide thepatients with the best possible treatment.
The objective of this chapter is not to advertise or to explain the use of the bone filler agents or themembranes in current use, globally. It is intended to introduce the theory of alveolar bone regeneration
therapy for the success of the implant treatment, to provide the readers with accurate clinical scientificknowledge.
II. Alveolar bone regeneration therapy

There are three factors that cannot be forgotten when performing bone regeneration therapy. The
osteogenicity, osteoinductivity, and osteoconductivity (Fig 1-2-1). In theory, the presence of these threefactors should result in bone regeneration. Osteogenicity indicates the presence of the cells that are able
to form the bone. For example, bone fragment is extracted with the bone forceps from the maxillary
tuberosity to be used as bone filling agent. The maxillary tuberosity is mainly cancellous bones thatconsist of osteocytes and bone marrow stromal cells. Here, the bone filling agent becomes an own bonewith a high osteogenic ability. Osteoinductivity refers to the property that can induce the bone marrowstromal cells that reside in the host bone or allografted bones for a specific differentiation of theosetocytes. The bone marrow stromal cells are undifferentiated mesenchymal cells that have the ability todifferentiate into adipocytes, fibroblasts, chondrocytes, and myocytes in addition to osteocytes. However,with the activation of bone morphogenic proteins (BMPs), these set of cells can selectively differentiate
into osteocytes and achieve osteoconductivity. Such property is known as the osteoinductive abiliy, andthe typical components are BMP-2 and BMP-7. The osteoconductivity refers to the property of thematerial to provide an environment where the cells can proliferate and differentiate to secrete thesubstrates. In short, it is the ability of the material to accommodate the cells acting as a scaffold for thecells to proliferate and divide, or construct blood vessels, accordingly.
Take a blood clot in an tooth extraction socket as an example. The blood clot formation is the initial step,and the most important of the wound healing /bone regeneration process. The blood clot is constructed of
the complex mesh of fibrins. In the interior of the blood clot, platelets can be found to be activated tosecrete cytokines of significant variation. Amongst these cytokines, there are those with a role to draw thestromal cells from the surrounding bone marrows towards the extraction cavity; those that facilitate theproliferation process; or those that attract the vascular cells for capillary constructions. The
undifferentiated stromal cells that have been accumulated by the cytokines proliferate further anddifferentiate within the blood clot made up of the fibrins to extend the bones. Therefore to summarize,
osteoconduction is the responsibility of the fibrin mesh, and the osteoinduction is orchestrated by thecytokines secreted by the platelets.

Fig.1-2-1 Three factors of osteoanagenesis
III. GBR and membranes
The extracted socket becomes filled with blood clots, which eventually becomes covered with the gingivae.
The internal structure of the jaw is undergoing continuous osteogenesis for the extraction socket to behealed, but it is often found in the clinical settings that the healing of the socket is associated with thecollapse of the bone structure. Two reasons can be thought as the cause for this phenomenon. First, therate of healing of the gingivae is faster than that of the osteogenesis, therefore the gingivae structure
invading the internal structure of the extraction socket results in the prevention of the osteogenic process.
Second, the blood clot constricts in the wound healing process and forming the indentation itself.
Whichever the order, the cause is the gingivae invasion into the space that is required for the extension of
the bone. Therefore by implementing a method to maintain this space by preventing the gingivae
invasion, as much bone augmentation can be possible. The solution to this problem for both “exclusion of
the soft tissues”, and “preservation of space” is the use of membranes, and the theory for this wasestablished by Nyman in 19821). In their method, they inserted the membrane with the aim to regenerate
the periodontal tissues, and termed it periodontal guided tissue regeneration (GTR), but the membrane
applied for bone augmentation/ bone regeneration, this should be referred to as guided bone regeneration(GBR). This technique was discussed with the extraction socket healing as an example, but thefundamentals of this technique “the exclusion of the soft tissues and the preservation of the space canfacilitate the osteogenesis” is applicable and one that is essential for all of the bone augmentation/
regeneration methods, and one that should be the foundation for success of the GBR method to facilitate
the implant treatment.
An ideal membrane is one that has the following properties:
1) Processibility, such that it can be trimmed with ease and be applied to even the complicated surgicalsurfaces.
2) Promote the formation of a stable blood clot3) Support the attachment of the flap (gingival flap)
4) Has the resistance to modification during its function in the oral cavity for the preservation of space
5) Retain these properties till the healing process of the tissues is complete6) One that can be removed easily
The membrane materials can be largely divided into those that are nonabsorbable and absorbable. The
Millipore filter and the expanded polytetrafluoroethylene (ePTFE) membrane are classed under the
nonabsorbable types. ePTFE membrane consists of a titanium frame, designed specifically with the aim
to “preserve space”. The non-absorbable membrane is characterized with a high biocompatibility and withthe ability to maintain its function within the physiological settings for a long time. In addition to theseproperties, the titanium frame of the ePTFE membrane is specialized with its ability to preserve theamount of space. The drawback to the non-absorbable type is the necessity of the secondary surgery to
extract the membrane, and the incidence of exposure of this type has been reported to be higher than its
counterpart 2).
Absorbable membrane can be further divided into those composed of synthetic polymers or collagens,where Type I is the most prominent or there are others that consist of both Type I and Type III. The raw
materials used are usually of the animal origin such as the skins and tendons of pigs and cows. The
collagen membranes are able to be digested by the endogenous collagenases within the body eventually,
but remain functioning for at least a month, and up to half a year in some types. The synthetic polymer
membranes include polylactic acid (PLA) polymers, Polyglycolic acid (PLA/PGA), and Polyglactin-910(polyglactin-910). These synthetic polymer types undergo hydrolysis within the body to be absorbed

eventually, but remain functioning for few months to half a year. The absorbable membranes, especiallythe collagen type, are highly compatible with the biological tissues and attach the flap well therefore canprevent the exposure of the membrane. In addition, since it is absorbable, in the body, the secondary
surgery for the extraction of the membrane is not required. On the other hand, the absorption rate of themembranes varies amongst the individuals therefore it is not possible to accurately determine the timethat it remains in function. Another disadvantage is that with this type lacks in the strength thereforebone filling agents may be required to preserve the space required.
There are a variety of membranes, as have been mentioned here, but the main types applied under theclinical settings involve collagen membranes and ePTFE membranes.
The comparable studies that have investigated the differences in outcomes in the application of thecollagen membrane and the ePTFE membranes for bone augmentation in the cases of bone fission, theresults indicated that there were no differences in their abilities to facilitate bone augmentation ion thosewhere there were no exposures of the membranes. Where exposures did occur, it was found that collagenmembrane to be the more suitable of the two types 3).
The application of membranes in implant treatment is usually for bone augmentation/ regeneration
purposes, but can also be used as the recovery measure in situations where the schneiderian membranecomes unstuck during sinus-lift procedures (Fig. 1-2-2). The membrane must be the absorbable type inthis case. Ensure a complete cover of the perforation with the absorbable membrane, and stabilize it withthe application of the bone filling agents before closing the flap.

Fig. 1-2-2-a to e. A case where the perforation of the schneiderian membrane was closed with theapplication of the absorbable membrane:
a. Sinus-lift procedure was initiated with lateral fenestration surgery.
b. A large perforation resulted at the elevation stage of the schneiderian membrane (the black
region at the 12 o’clock position is the perforated region).
c. Covering the perforation with absorbable collagen membrane.
d. Bone filling agent was added on the top, with the placement of the membrane.
e. The localization of the bone filling agents to underneath the sinus mucosa can be observed in
the radiograph.

IV.

Importance of “space preservation”

The preservation of secure space is the key to a successful outcome of bone augmentation with
GBR technique. In comparing the clinical outcome of the ePTFE that have been strengthened
with the titanium frame and that without, those with the adjunct has a better outcome 5). This isowing to the ability of the titanium frame to protect the space required for the boneaugmentation. Other recommended means to secure the preserved space are to use bone screws
and bone tack to stabilize the ePTFE membrane. Provided that the application of membrane
itself is sufficient to achieve adequate bone augmentation/ regeneration, there is no need for the
adjunct tools, however, the reality is that the blood clot constriction, and the exertion of pressure
from the membranes make this difficult to accomplish this. In particular, as in the caseillustrated in Fig. 1-2-3, the even the force applied for suture can be enough to exert pressure on
the membrane and restrict the amount of space possible. For this reason, it is often
recommended to support the membrane with the addition of the bone filling agent in order togain sufficient bone augmentation/ regeneration. Given that that bone filling agent itself canpromote the bone augmentation process, or more specifically, the higher the osteoconductivity
and osteoinductivity, a satisfactory bone augmentation and regeneration can be accomplished.

Fig.1-2-3
It is difficult to secure the amount of space required with only a membrane and blood clot, incases such as this, thus it is desirable to pack bone graft substitutes.
V.

Bone filling agentsThe requirements for materials to be used as bone fillers:
1) Highly biocompatible2) Stabilize blood clot and preserve the space
3) Has osteoconductive and osteoinductive abilities that support angiogenesis, proliferation of
the bone marrow stromal cells and division of osteoblasts4) Become a part of the host bone structure by its degradation
The bone filling agents can be largely separated into alloplast, xenograft, allograft and autograft
types. The bone filling agents, with the exception of the own bone, have been processed into
granules to have the ability to adopt to fill even the complex bone deficient structures. They have

also been referred to as the granular bone graft materials. The sizes of these particles vary fromthose that are within 100 to 300 μm diameters up to those of 300 to 1000μm diameters. The
smaller the diameter of the granules, the larger the surface area can be used, and the larger the
diameter of the granular graft particle, the slower the degradation.
A. Alloplast
There are absorbable and non-absorbable types amongst the alloplast materials. Bioceramics, β- tricalcium phosphate (TCP), hydroxyapatite (HA), calcium sulfate, calcium carbonate calcium
carbonate (derived from corals), and calcined gypsum are examples of the absorbable type, andporous HA, high-density HA, bioactive glass, and hard tissue replacement polymer (HTR
polymer) (based on polymethyl methacrylate (PMMA)) are the non-absorbable types. The
biocompatibility of the alloplasts reduces its risks of infection derived from the material, andwith the added advantage of the manufacture in large quantities, makes it a viable option;
however there have not been many reports of successful outcome with its application for the
GTR/GBR procedures. Furthermore, its osteoconductive ability is not one that is superior to theother bone grafting substituents. Application of alloplasts alone for GTR/GBR is notrecommended, at present.
B. Xenograft
Xenografts are bone graft substituents of other animal origin, where in the field of dentistry uses
bones from cows as the raw material. The proteins are decomposed by undergoing heat andchemical treatments, then are processed into the granular shape without any modifications tothe structures of inorganic materials. As the structure of the inorganic matter remains as thenatural HA, it provides with an osteoiconductive environment that is more suitable for theosteogenic process. The bone graft material acts as the initiator of the neonatal bone formation,
and a significant amount of time is required for the absorption of the material 6).
A type of xenograft material that has coated the natural HA with a synthetic peptide (P-15) isalso present. The Type I collagens act as a natural cell scaffold with a specialized peptide
structure, and the P-15 has been synthesized to mimic this structure. The theory here is that the
P-15 coat should improve the compatibility of endogenous HA with the other cells. These bone
graft material use the bone of the cows as the raw material, but has been reported to be devoid of
the prion proteins that induce Bovine spongiform encephalopathy (BSE), and therefore with norisks of its infection 7), 8).
C. Allograft
The allograft uses the human bone that has been donated to the tissue bank. Since the raw
material is of the same species, its similarities in their basic organic and inorganic structures canbe applied directly to those of the host site, making this type of graft effective for osteogenesis. In
the United States, allograft transplant had been applied to over a million cases by 2004. There
are decalcified freeze-dried bone allograft (DFDBA), freeze-dried bone allograft (FDBA), andsolvent-treated human HA to this type. These bone graft materials have excellent
osteoconductive and osteoinductive abilities. In the 1990s, a study to quantify the amount of

BMP contained in DFDBA was conducted which reported that the amount of BMP contents
differed significantly according to the age of the donor and the treatment method that
subsequently led to the disparity in the osteoinductive abilities. The present belief however isthat the amount of BMP contained in the DFDBA is not sufficient to precipitate osteoinduction,questioning the ability of the allograft material with its osteoinductive ability. The FDBA havenot undergone decalcification therefore requires a longer period of time for its absorption into thebody than with the DFDBA. Meanwhile the solvent-treated human HA has been treated via thetutoplast method without either of the decalcification nor freeze-dry processes. This tutoplast
treatment is thought to retain the original organic and inorganic structures, therefore havestrong oseteoconductive ability to promote the osteogenesis. The DFDBA, FDBA, and human HA
are widely used in clinical settings with their efficacy proven (Fig.1-2-4). In addition to the
granule-forms, block-forms are also present that are able to be used in place of chin or ramus
grafts.
The one factor that should be considered is the risk of infection from the bone graft material.
Theoretically, the transmission of infection is possible even after the material have undergone
thorough screening and disinfection treatments. The risk of transmitting HIV infection has beenreported to be 1/8,000,000 and risk lower than this.

Fig.1-2-4-a to f. A case where the extraction socket preservation method on the anterior maxilla
was conducted with absorbable collagen membrane.
a. A large amount of abnormal granulation tissues in the extraction socket and the apical areab. The abnormal granulation was removed completely, which revealed thin labial alveolar
crest. By closing the gingival flap without the any treatment, the aesthetic outcome with an
implant treatment would become difficult due to the indentation on the labial side of theanterior maxilla.
c. Trim the membrane to be inserted under the palatal gingival flap, and upon determining thelocation, fill the extraction socket and its surroundings with the human HA.
d. Cover the bone graft material with the membrane, followed by the gingival flap.
e. Relax the gingival flap before closing with absorbable suture.

f.

A significant amount of regenerated bone could be found half a year later.

D. Autograft
Autograft refers to the graft of own bone, usually extracted from other parts within the oralcavity, iliac bones or fibulas. The safety and its attachment ability are both effective on top of
their osteogenic, osteoconductive, and osteoinductive abilities.
The bones from the intraoral cavity can be obtained from maxillary tuberosity, mandibular torus,
buccal sides, and mental region, but those extracted from the maxillary tuberosity are high inosteoblasts and bone marrow stromal cells, therefore is the most suitable for GBR. Autograft is
the gold standard of bone graft material with its numerous advantages but its drawback is thelimited amount of bone quantity that is available. For this reason the combination of allograft
and the xenografts are often used in the clinical settings since it is not recommended to combine
the autograft with the other graft materials. When applying autograft, it should be spread to anarea of bone or the exposed section directly, then spread a layer on top of this. The bone
fragments that were extracted from the implant surgery must be preserved in the physiologicalsaline solution till the end of the surgery (Fig. 1-2-5). If the GBR is required to mend the bonedehiscence resulting from implant surgery, this should be treated with the autograft.

Fig. 1-2-5 a to d.
a. The drilling for the implant cavity results in the bone fragments to come attached to thedrill blade. Limit the water infusion and avoid suction of the bone fragements.
b. Remove the bone fragments with the perio-probe, and preserve the fragments in thephysiological saline solution to prevent it from dryingc. Own bone that has been obtained with the 2 mm, 2.5 mm, 3.2 mm and 3.7 mm drill
blades.
d. A sufficient amount of graft material for a small area of bone deficiency after the implantinstallation.
VI.

New generation of bone graft agentsThe new generations of bone graft materials are biomaterials that have encompassed the idea oftissue engineering, and this is thought to evolve as the main stream therapy. The growth factors
play a major role in this process (Fig. 1-2-6). The growth factors act as the source ofosteoinductivity, and attract the undifferentiated stromal cells, stimulate its proliferation and its
differentiation. The growth factors are not included in the bone graft materials except in those of
the autograft agents. Platelet rich plasma (PRP) and plasma rich in growth factors (PRGF) havedrawn attention in the recent years owing to their high level of growth factors included in thesematerials. In Japan, the research into the application of fibroblast growth factor-2 (FGF-2) in theregeneration of the periodontal tissues has been conducted extensively.
A. BMP-2 and collagen sponge
Bone morphogenic proteins (BMPs) are a potent inducing agent of osteogenesis. BMP-2 andBMP-7 have been particularly focused due to their success in the treatment of intractablefractures in United States and Australia. It is a recent development that BMP-2 has been
approved for its use in bone regeneration therapy (GBR and sinus lift) in the field of dentistry. It
is a medicinal product that has not yet been approved in Japan. The method for application ofthis agent is to add to the collagen sponge to the area of bone augmentation/regeneration. BMP-2
and collagen sponge are both synthetic materials therefore have no risks of infection.
B. PDGF and β-TCP
Platelet derived growth factors (PDGF) plays important roles in wound healing, angiogenesis,
cell proliferation and regulation of osteocytes, and a large number is contained in the platelets.
The PRP which is the blood plasma enriched with platelets contain 5 to 10 times theconcentration of PDGF, whereas in the Gem 21 (growth factor enhanced matrix-21) contains
1000 times that of the PRP. This is highly concentrated PDGF is applied in a mix with thealloplast, β -TCP. Its application to the regeneration of the periodontal tissues has been
approved, but this has not been applied in Japan.
VII.

Quality of regenerated bone

There have always been conflicting views in the long-term preservation of the implants with thebone that had undergone regeneration processes in the same way as the original, innate bone.
The recent findings have reported that the survival rate of the implants over five years were
found to be on average 96% with the bone that had been regenerated with GBR technique,therefore confirming there to be no significant difference between those that have been
implanted to the innate bone.
VIII.

Final word

Many different kinds of membranes and bone graft materials have been developed, but the key toa successful outcome with the GBR technique is thought not to be in the selection of thematerials, but in the complete closure and the healing of the area operated. In case of themembrane exposure, it can also contaminate the bone grafting material, leading to the worsened
state of the bone. The meta-analysis conducted by Machtei showed that the exposure ofmembrane in GTR method does not lead to a huge failure but the membrane exposure in GBR
method have led to a significant setback. The average growth of the bone without the membrane
exposure was indicated to be 3 mm, whereas the growth of 0.5 mm was noted where themembrane exposure did occur 11).
The key factors for a complete closure of the gingival flap and its healing have been listed asfollows:
1) Conduct a tension-free closing of the gingival flap, having fully released the structure.
2) The incision should be formed that is widened at the bottom to ensure sufficient supply ofblood.
3) Apply horizontal mattress suture, and draw in the gingival flap as a whole structure.
4) Avoid overloading the bone graft agentsThe alveolar bone regeneration is an area of particular interest from the clinical setting as well
as research. A number of scientific findings have been derived from the clinical studies applying
membranes and bone graft materials. The main emphasis has been placed on the application of
the growth factors and in the development of scaffolds that are highly osteoconductive (Fig.
1-2-6).
Using animal subjects, trials for the application of the growth factors, which have been inducedwith the genetic engineering, into scaffolds for its transplantation to the host site are being
conducted. It is an area that can expect a great development hereafter, and the future of
implants is optimistic.

Fig.1-2-6 Three factors of the new generation of bone regeneration.

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